I. Field of the Invention
The present invention relates generally to the treatment of electrical injury. The invention is directed to methods and compositions for the treatment of cell membrane damage and tissue injury caused by the disruption of cell membrane integrity following electrical injury. In particular, the invention concerns the treatment of tissue damage and the enhancement of cell survival through the sealing of permeabilized cell membranes by administering an effective amount of a composition comprising a surface-active copolymer, and more preferably, through the co-administration of a surface-active copolymer and a high energy phosphate compound.
II. Background of the Invention
A high percentage of major electrical trauma victims suffer extensive tissue necrosis, high-level amputations and become permanently disabled (DiVincenti et al., 1969). Among electrical utility workers in the United States, the majority of shock victims experience hand-to-hand or hand-to-foot contacts between 6 and 10 kv. Electrical shock simulations by computer suggest that with perfect electrical contacts such circumstances can produce electric field strengths in upper extremity tissues ranging between 60 and 160 V/cm (Tropea & Lee, 1992). Fields of this magnitude can produce skeletal muscle and peripheral nerve membrane damage through electroporation (Lee & Kolodney, 1987b), Joule heating (Lee & Kolodney, 1987a; Lee et al., 1988), or a combination of both.
Skeletal muscle and peripheral nerve necrosis appears to be the primary cause of the high amputation rates associated with electrical trauma. In cases of high-voltage electrical trauma, loss of structural integrity of the cell membrane is believed to be a central pathophysiologic event (Lee & Kolodney, 1987a; Lee & Kolodney, 1987b; DiVincenti et al., 1969; Tropea & Lee, 1992). Membrane damage is often manifested by the release of intracellular contents into the intravascular space, which is, indeed, one of the clinical hallmarks of major electrical trauma. It has been postulated that in the majority of electrical injury victims, cell membrane permeabilization is the most important event in these necrotic processes (Tropea & Lee, 1992; Bhatt et al., 1990; Jaffee, 1928).
`Membrane permeabilization` is the production of discrete openings at numerous sites in cell membranes. The consequences of membrane permeabilization are numerous, and include loss of cytoplasm and some of the contents thereof, disruption of ionic concentration gradients, and depletion of intracellular energy stores. Cells which suffer cell membrane permeabilization may thereafter die and the tissue will subsequently undergo necrosis. One of the consequences of the permeabilization is concomitant egress of the contents of the cell, and without some means of potentiating the repair of the openings, cell survival rates can often be unacceptably low.
One of the more serious consequences of cell membrane permeabilization is the significant depletion of intracellular energy stores. Under normal circumstances, cells maintain a high level of ATP by using oxidizable substrates as sources of free energy. Following permeabilization, a considerable amount of cellular energy would be expended in an attempt to maintain intracellular ionic balances despite the efflux of ions through the permeabilized cell membrane, according to their concentration gradients. During this period of imbalance, the ATP demand will increase to fuel the repair processes, but the intracellular reactions which regenerate ATP stores will be inhibited, leading to a further depletion of the cellular ATP stores. This can render the cell unable to re-establish appropriate ionic gradients across the membrane, prevent it from functioning normally, and ultimately lead to cell death.
A system in which such a depletion of ATP has been well demonstrated is in animals suffering hemorrhagic shock. It has been demonstrated that administration of ATP-MgCl.sub.2 before, during, and even after a period of severe shock in rats had a beneficial effect on the animals' survival. The ATP was administered along with the MgCl.sub.2 in order to prevent chelation of divalent cations from the vascular system by ATP administered alone. Furthermore, MgCl.sub.2 inhibits the deamination and dephosphorylation of ATP. Thus, by administering equimolar amounts of ATP and MgCl.sub.2, a higher concentration of ATP will be available to the tissues than if the ATP were administered alone. The results of this study suggested that the beneficial action of ATP-MgCl.sub.2 may not have been through vasodilation alone, and it was postulated that the administered ATP could have a "priming effect" on the intracellular synthesis of ATP (Chaudry et al., 1974).
The actual method of cell membrane repair in vivo remains unknown, although researchers have made some inroads toward understanding the mechanisms involved. Calcium ions have been implicated, through both in vitro and in vivo studies, as having a critical role in membrane fusion and repair (Aldwinckle et al., 1982; Papahadiopoulos et al., 1990; McNeil, 1991). Membrane and cytoskeletal proteins, including spectrin, dystrophin, and actin may also be actively involved in the maintenance and repair of the cell membrane in vivo (McNeil, 1991). It has also been suggested that chemical factors may play a signal-like role in wound healing at the cellular level (McNeil, 1991).
Unfortunately, no immediately effective method for treating such injuries currently exists. Also, although methods have been described which reduce the deleterious effects of cell membrane permeabilization, each suffers from particular limitations. The addition of serum to cells permeabilized through the use of electrical pulses has been shown to enhance cell survival (Bahnson & Boggs, 1990). However, serum proteins do not effectively reach damaged cells in vivo. Accordingly, there is a currently lack of a safe and effective method of treating the tissue damage which results from electrical injury.
As membranes form spontaneously when surfactants (amphiphiles) are mixed in an aqueous solvent at sufficient concentration, the inventor hypothesized that it may be possible to seal damaged cell membranes by exposing them to adequate concentrations of a non-cytotoxic non-ionic surfactant.
Several biomedical applications of surface active copolymers, and in particular poloxamers, have been described. These include use as an agent in the preparation of stable and concentrated antiserum, as an emulsifying agent, as a wetting agent in an antiseptic skin cleaning formulation (Rodeheaver et al., 1980), as an enhancer of drug or antibiotic levels in the blood, and as a tool in the study of tumor metastasis (Schmolka, 1977).
Specifically, poloxamer 188 has been used as an emulsifying agent since the 1950s. Initially it was used as a surfactant to protect red blood cells in the membrane oxygenators of early model cardiopulmonary bypass machines, and was shown to prevent hemolysis and lipid embolism. It has been used as an emulsifying agent in foods, oral drugs and cosmetics and is an FDA-approved food additive. Poloxamer 188 has been shown to block the adhesion of fibrinogen to hydrophobic surfaces and the subsequent adhesion of platelets and red blood cells. It is currently an FDA-approved surfactant in the synthetic blood replacement flusol (Check & Hunter, 1988; see also U.S. Pat. Nos. 4,879,109; 4,897,263; and 4,937,070; each incorporated herein by reference.
As mentioned above, there is a particular need for a safe and effective method of treating electrical injury victims. Because cell membrane permeabilization is an important factor leading to tissue necrosis after electrical injury, this suggests that effective therapy for victims of electric shock should re-establish cell membrane structural integrity. A method which addresses the problems of permeabilization and energy store regeneration associated with such an injury would be a particularly novel and advantageous development in this field.